Infection by human papillomaviruses (HPV) at specific epithelium cells to induce epithelial proliferations plays an important role for cervical carcinogenesis. About 99 percent of confirmed cervical cancer cases are found to be associated with HPV infection with biopsy-confirmed squamous intraepithelial lesions (SIL) or cervical intraepithelial neoplasia (CIN). The incidence of HPV infection, primarily transmitted through sexual contact, is highest among young women and about 20 millions of sexually active men and women worldwide are currently infected. Approximately 1% of the population has genital warts and 4% of women have cervical precancerous lesions, such as low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL) or atypical squamous cells of undetermined significance (ASCUS).
These lesions are preferentially observed in women aged 35-40 yrs and have a high risk of progressing towards invasive cervical cancer. It is generally thought that persistent infection of human papillomavirus (HPV) is essential for developing precancerous epithelial lesions. Infections of high-risk types of HPV in women with LSIL may or may not progress to HSIL. In fact, remission occurs in the majority of LSIL human subjects while some progress to HSIL. Although 99.7% of cervical cancers are HPV positive, the integration of viral genome into the host genome is required to facilitate the necessary genetic expression for developing into HSIL or cancer. In fact, only one in every 10 women with persistent HPV infection may develop higher grades of CIN lesions, such as cervical intraepithelial neoplasia (CIN) grade 2 and grade 3 (CIN2, and CIN3, respectively), and a portion of these epitheliual lesion cases may ultimately progress into cervical cancer.
In the past, screening for cervical cancer has been based on conventional cytology screening tests, e.g., obtaining papanicolaou (Pap) smears for cytological staining tests, and suspicious smears are followed up with colposcopy, and/or histological biopsy. The use of these cytological screening tests contributes to a reduction in the mortality of cervical cancer. However, due to subjective test criteria, there are various drawbacks for pap smear tests: difficulty in obtaining samples, poor inter- and intra-observer agreement, high rates of false negatives and false positives, requiring specialized labs staffed with highly trained personnel, and inability to identify the majority of HPV-infected human subjects. More reproducible assays are needed to improve the current screening tests and to avoid unnecessary medical intervention and psychological distress in affected women. The current conventional cervical cytology screening tests have sensitivity varied from about 30% to about 87%.
Detecting HPV infection by nucleic acid tests, such as “DNA Hybrid Capture”, has been developed with high assay sensitivity, but is still not ideal, due to not only its high cost, assay operation procedures, the requirements for facility, equipment, and highly trained personnel, but also its very low positive predictive value (PPV) in cervical intraepithelial neoplasia (CIN) testing samples. Assays like PreTect HPV-Proofer® provide the detection of E6/E7 mRNA with sensitivity equivalent to HPV Hybrid Capture tests with higher positive predictive value but cannot directly detect E6/E7 oncoprtoeins in situ. In addition, DNA testing could not differentiate disease stages after HPV infection nor the diagnosis of different cell lesions (e.g., cannot differentiate LSIL from HSIL, nor CIN lesions from non-transforming latent or remissive viral infection). What is needed is a low cost, simple, sensitive and specific assay that can be routinely performed in a clinical lab or doctor's office and is capable of detecting early stages of epithelial lesions, distinguishing LSIL from HSIL, and/or predicting the risk of progression into cervical cancer.
Known protocols for producing monoclonal antibodies are generally unsuitable for the production of anti-HPV monoclonal antibodies and cannot be used in immunocytochemical diagnostic tests performed on human subjects of the general population. This is because antibodies produced by these protocols will not necessarily react with the naturally occurring HPV viral proteins in infected human cells. It is thought that the epitopes recognized by antibodies if generated by conventional protocols will not necessarily be those epitopes which are resistant to the harsh procedures involved in standard sampling, fixing and storing of clinical specimens. In addition, three problems exist in clinical HPV detection. One is that HPV proteins in clinical samples are present in very small quantities. Secondly, there are too many HPV types and most HPV types present in clinical samples are not known or systemically identified due to the lack of available antibodies. Third, HPV virus can not be cultured in labs by standard tissue culture techniques. Thus, there are no available HPV proteins purified in large quantities as immunogens for generating anti-HPV antibodies, and there are no available HPV proteins or purified anti-HPV antibodies to recognize anti-viral antibodies or viral proteins present in clinical samples for clinical HPV detection.
Only about 15 HPV types out of more than 100 HPV types are at high risk of developing into cervical intraepithelial neoplasia (CIN) or cervical cancer. Among them, around 70% of reported cervical cancer cases and 50% of reported CIN 2 and CIN 3 cases are caused by two high risk HPV types, i.e., HPV type-16 and HPV type-18. However, some progressive cervical cancer cases are reported to be associated with low risk HPV types, and infection of some high risk HPV types will never progress into cervical cancer. Infections by these two prevailing high risk HPV types do not correlate with tumor development or cancer progression. For this reason, it seems important to identify those HPV-infected human subjects that express particular oncogenic proteins rather than just identify HPV infection by high risk types.
Thus, there is a need for detecting the expression of HPV-related oncoproteins in clinical samples as these oncoprotiens may serve as cervical cancer biomarkers to better predict the risk of developing high grade of cell lesions or cervical cancer-related diseases. There is also a need to develop anti-HPV antibodies and appropriate HPV immunoassays to detect the presence of invasive cervical cancer and/or HPV-related oncoproteins as cervical cancer biomarkers and predict the risk for malignant transformation of epithelial lesions into cervical cancer.